Up to date concepts about Von Willebrand disease and the diagnose of this hemostatic disorder.

Abstract The authors review the current data in literature regarding the recent knowledge about hemostase, coagulation and clinical and laboratory diagnostic algorithms of hemostatic disorders. They also present the pathological classification of bleeding disorders - the basis to clinical approach of these diseases. Abbreviations: AD=autosomal dominant; Ag=antigen; DNA=deoxyribonucleic acid; ADAMTS13=serum metalloproteinase; AR=autosomal recessive; Arg=arginine; RNA=ribonucleic acid; VWD=von Willebrand disease; Cys=cysteine; C1—C9=factors of the seric complement; ELISA=enzyme linked immuno assay; FI---FXIII=plasmatic factors of coagulation; Glu=glutamines; Pg=platelet glycoprotein; HMW=high molecular weight; IL=interleukin; SLE=systemic lupus erythematosus; Met=methionine; PFA=automated study test of platelets aggregation; RCo=ristocetin cofactor; RI PA=ristocetin induced platelet aggregation; Tyr=tyrosine; VWF= von Willebrand factor


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The diagnosis of hemorrhagic syndromes Clinical examination
The diagnostic approach of hemorrhagic syndromes starts with the clinical evaluation of the child. The first differentiation assigns the bleedings to a local cause or a systemic affection. From this point of view, it is important to find out the answer to the previous requests of hemostasis (the ligature and the sectioning of the umbilical cord, detachment of the umbilical blunt, dental eruption, dental extractions, surgeries, menarche, etc.).
The usual bleeding area also gives the diagnosis guiding elements: • Cutaneo mucous hemorrhages (bruises, purpura, epistaxis, gums hemorrhages, menorrhagia, etc.) are especially met in the anomalies of primary hemostasis (thrombocytopenia, thrombocytopathia, von Willerbrand disease, etc.). • "Spontaneous" hemarthrosis and profound hematomas of the soft tissues are characteristic to coagulopathies. The timing of the appearance of hemorrhages according to the shutter traumatism represents a differentiation factor: bleedings with immediate post-traumatic start are characteristic for vasculo-platelet anomalies, while the hemorrhages with a late start compared to the moment of the trauma are met in coagulopathies.
The answer of the hemorrhages to the usual hemostatic measures (tamponade, suture of the wounds, vessels ligature, etc.) is also different in the anomalies of primary hemostasis -in which we obtain a clinically satisfactory answer compared to the coagulopathies in which only the substitution of the deficient factors of coagulation leads to the control of hemorrhages. The drugs administered to the patient in the past and in the present can affect the hemostasis.
A last aspect which can favors important information for the diagnosis is the familial history of hemorrhagic diathesis; this way we can answer the question whether the hemorrhagic syndrome is constitutional (hereditary, familial) or acquired.

The laboratory screening of hemostasis
The examination of the patient continues with the hemostasis laboratory screening investigations, which will allow the objective placement of the patient in one of the major category of hemorrhagic syndromes or which will allow even the diagnosis of the affection in some cases.
Although at present there are various laboratory tests for the investigation of hemostasis, there is no consensus regarding the tests that are unanimously used in the screening of hemorrhagic syndromes, especially preoperatory. Table 2 offers an inventory of the hemostasis screening tests that are used most often. Table 2. The hemostasis screening tests [2,3,4] The differential diagnosis compared to the results of the hemostasis screening tests is presented in Table 3. Table 3. The classification of the hemorrhagic syndromes according to the results of the coagulation screening tests [2] • Factors VIII, IX and XI deficits (The factors XII, PK/K, HMWK deficits, just like the lupus antigoagulant can prolonge APTT, but are not tipically associated with the hemorrhagic diathesis). The drugs administered to the patient in the past and in the present can affect the hemostasis; the anamnesis must be systematically oriented towards this aspect too. Moreover, the anamnesis regarding the organic affections which can affect the hemostasis (liver diseases, liver and renal neoplasms, infections, etc.) must be done.
A last aspect which can favor important information for the diagnosis is the family history of hemorrhagic diathesis; this way we are able to answer the question whether the hemorrhagic syndrome is constitutional (hereditary, familial) or acquired.

The confirming and analytical laboratory tests
These tests lead to the final diagnosis of the hemorrhagic disease and are generally the attribute of the specialized laboratories, which need technology and clever techniques, specialized laboratory personnel and are expensive. Their implications are obvious in cases of diagnostic difficulties, as in research, due to not being widely used in the usual clinical laboratories. These investigations also need specialized algorithms both in the execution and in their interpretation.

Von Willebrand disease The structure and the functions of von Willebrand factor
VWF is an adhesive glycoprotein, present in the plasma in a concentration of 5-10 mcg/ml. A part of the VWF molecules circulate in the plasma complexed by f.VIII; a high active form is stored in the secretory granules of the platelets and in the endothelial cells. The moment these cells are activated by the tissue lesion (ex. by contact with thrombin), they instantaneously mobilize the stored VWF.
It is a big-size protein made up of many subunits, connected covalently by disulphide bonds. It contains numerous bonding sites for the platelets and the basal membrane of the blood vessels. It is made up of 2813 amino acids, having a molecular weight of D = 309.000; it also contains N -places and 0 -glycosylated. A propeptide of great dimensions (741 amino acids) is attached to the mature VWF. The propeptide is identical to AgVW FII and plays an important role in the assembly and processing process of VWF multimers.
The great forms of VWF assembled in endothelial cells are processed in the plasma in forms of little dimensions by the action of a plasmatic metalloproteinase, ADAMTS 13.
In the plasma, VWF exists in the form of a series of multimers with the molecular weight which varies between 0,5 (dimers) and 20.000.000 D (multimers of great dimensions). From an electronomicroscopic point of view, it has a filament form and demonstrates the presence of many subunits arranged in a "head-to-head" and "tail-to-head" configuration.
The multimers construction block is the dimer, connected to other similar subunits by disulphide bonds situated near the N -terminal end of the mature subunit.
Disulphide bonds localized near the carboxy terminal end connect the multimers.
The mature subunit has a molecular weight of approx. 270 KD and contains 18,7% hydrocarbons. The hydrocarbons that are N -and -O connected are grouped at both ends of the subunits.
All the cysteine rests (169, representing 8,2% of the amino acids content of the molecule) are involved in sulphide bonds intra-and interbonds. Two arginine-glycine-asparagine sequences (RGD) are present in Willebrand prefactor; one of them -also present in the mature subunit of VWF -takes part in the construction of the connection sites for GPII b/ III a platelet receptor.
VWF protein demonstrates the presence of some repeated homologous segments, designated from H to D. A superfamily of proteins with a similarity of sequence with A domains of VWF contains proteins associated with the extracellular matrix, cellular adhesion and hemostasis. The crystalline structure of A1 and A3 domains of VWF is remarkably similar to the structure of the Ist domain of α2 / β1 integrin.
VWF structure is presented in Fig. 2.

Fig. 2 VWF structure
Many functional domains have been identified in VWF structure (Fig. 2): • major places, physiologically active, of bonding the fibrillar collagen situated in H3 domain; • At least two heparin bonding places which can interact with the heparin-like molecules in the structure of the basal membrane of the blood vessels; Together with other adhesion platelets, such as fibrinogen, fibronectin and thrombospondin -VWF interacts with GPIIb/ IIIa on the activated platelets contributing to the platelet aggregation (the secondary aggregation induced by agonists); • in VWF -factor VIII complex, factor VIII circular stabilizes, preventing its enzymatic destruction; in the absence of VWF, plasmatic T/2 of circulating f.VIII is more diminished, which proves the implication of VWF in coagulation.

VWF gene
VWF gene is situated, in humans, on the short arm of 12/12p chromosome). It is a large dimensions gene (178 Kb, 52 exons, representing approximately 0,1% of the DNA of chromosome 12).
There is also a non-functional, partial duplication of the gene (VWF pseudo gene) situated on chromosome 22. It represents the duplication of the middle side of VWF gene, from the exons 23 to 34, including the intervening sequences. It has a homology of approximately 97.2% of the authentic gene and is only present in humans.
The structure of VWF gene is presented in Fig. 3.   Fig. 3 The structure of von Willebrand gene -specific mutations to different subtypes are shown in the image [3,5] VWF biosynthesis It is limited at the level of the endothelial cells and the megakaryocytes; VWF is initially synthesized as pre-VWF monomer.
At the level of the endoplasmic reticulum of the endothelial cell, the transcription of the DNA takes place, producing a VWF RNA; the translation in RNA also takes place at this level. The dimmers are transported in Golgi apparatus.
At the level of Golgi apparatus, the N-linked hydrocarbonate is being processed, the O-linked glycosylation of the molecule and its sulfation. Moreover, the multimerization through disulphide bridges under the disulphide-isomerase, in acid environment is also initiated.
The Post-Golgi multimerization (favored by the presence of propeptide) and the propeptide cleavage is continued.

VWF storage and secretion
The storage is represented by the endothelial pool, at the level of Weibel-Palade corpuses (together with angiopoietin 2, tPA and IL-8), whose origin is in Golgi apparatus and the platelet pool, at the level of α granules. In both situations, the stored molecules form tubular structures, electromicroscopically evidenced and having a diameter of 150 Å. A transmembranar protein -P selectin -is a component of α granules membrane and the Weibel-Palade corpuses. Its biosynthesis is restricted (just like the case of VWF) to endothelial cells and megakaryocytes.
The N-terminal domains (D1 -D3) are necessary for the storage of VWF; The selection of these domains leads to the constitutive secretion of VWF.
The secretagels are: thrombin, fibrin, histamine, C 5 -C 9 components of the complement, more bioactive lipids, for example sfingosine-1-phosphate and DDAVP for the endothelial cells.

The molecular genetics of hereditary VWD
There are two types of pathogenic VWD:

Fig. 2 VWF structure
• the quantitative deficit of VWF; it is realized through the deletion of a gene segment, or nonsense units which lead to the finishing of the synthesis of VWF protein by the insertion of a code stop in the coding sequence of VWF gene. • qualitative structural or functional anomalies of VWF, generally produced through punctual mutations/ the substitution of only one nucleotide in the coding sequence and, so, of only one aminoacid at the level of VWF protein. The quantitative deficit of VWF can be severe or light.
In the severe quantitative deficit of VWF (type 3 of VWF), VWF is undetectable or has very low vales in plasma or platelets.
In light quantitative deficit (type I von Willebrand disease), there are low circular levels of VWF. The responsible mutations have been identified in approximately 55% of the studied cases.

VWD history
In 1926, Erik Von Willebrand described a 5-yearold Finnish girl, from Aaland Islands, with a hemorrhagic diathesis which was different from hemophilia A through the AR transmission, the presence of the cutaneous hemorrhages (and not joint-related) and the prolonged bleeding time [1].

The laboratory examinations in hereditary VWD subtypes
The researches in the last years have demonstrated that different mutations present in hereditary VWD have similar effects on the structure and function of VWF, which has led to a simpler classification of the disease, based on the pathogenic mechanism ( Table 4).   Table 6. Represents a synthesis of the main clinical and paraclinical characteristics of hereditary VWD subtypes [2].